Human peroxisomal thioesterase

ABSTRACT

The invention provides a human peroxisomal thioesterase (PxTE) and polynucleotides which identify and encode PxTE. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of PxTE.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of ahuman peroxisomal thioesterase and to the use of these sequences in thediagnosis, prevention, and treatment of cancer, inflammation, anddisorders associated with fatty acid metabolism.

BACKGROUND OF THE INVENTION

Two soluble thioesterases involved in fatty acid biosynthesis have beenisolated from mammalian tissues, one which is active only towardlong-chain fatty-acyl thioesters and one which is active towardthioesters with a wide range of fatty-acyl chain-lengths. Thesethioesterases catalyze the chain-terminating step in the de novobiosynthesis of fatty acids. Chain termination involves the hydrolysisof the thioester bond which links the fatty acyl chain to the4'-phosphopantetheine prosthetic group of the acyl carrier protein (ACP)subunit of the fatty acid synthase (Smith, S. (1981a) Methods Enzymol.71:181-188; Smith, S. (1981b) Methods Enzymol. 71:188-200).

E. coli contains two soluble thioesterases, thioesterase I which isactive only toward long-chain acyl thioesters, and thioesterase II(TEII) which has a broad chain-length specificity (Naggert, J.et al.(1991) J. Biol. Chem. 266:11044-11050). E. coli TEII does not exhibitsequence similarity with either of the two types of mammalianthioesterases which function as chain-terminating enzymes in de novofatty acid biosynthesis. Unlike the mammalian thioesterases, E. coliTEII lacks the characteristic serine active site gly-X-ser-X-glysequence motif and is not inactivated by the serine modifying agentdiisopropyl fluorophosphate. However, modification of histidine 58 byiodoacetamide and diethylpyrocarbonate abolished TEII activity.Overexpression of TEII did not alter fatty acid content in E. coli,which suggests that it does not function as a chain-terminating enzymein fatty acid biosynthesis (Naggert et al., supra). For that reason,Naggert et al. (supra) proposed that the physiological substrates for E.coli TEII may be coenzyme A (CoA)-fatty acid esters instead ofACP-phosphopanthetheine-fatty acid esters.

CoA plays an important role in the synthesis and metabolism of fattyacids. Esterification of the fatty acid carboxylic acid group with CoAcreates a thioester bond which activates the fatty acid molecule fornucleophilic attack and subsequent metabolic conversions. Likewise,hydrolysis of the fatty acyl-CoA thioester bond renders the fatty acidcarboxylate group unreactive toward nucleophilic attack.

Peroxisomes are single, membrane-bound, spheroid organelles present invirtually all eukaryotic cells. The peroxisome matrix contains more thanforty enzymes which are involved in a variety of metabolic processesincluding peroxide-based respiration, synthesis of plasmalogen and bileacids, beta-oxidation of fatty acids, and glyoxylate transamination.Peroxisomal matrix enzymes are synthesized on free cytoplasmic polysomesand are imported into peroxisomes without subsequent proteolyticprocessing. Most peroxisomal enzymes contain a C-terminal SKL(ser-lys-leu) matrix targeting sequence.

More than half of the enzymes present in mammalian peroxisomes areassociated with lipid metabolism (Baumgart, E. et al. (1996) Proc. Nat.Acad. Sci. 93:13748-13753). Beta-oxidation of very long straight-chainfatty acids, branched-chain fatty acids, dicarboxylic fatty acids, andeicosanoids occurs within peroxisomes. Beta-oxidation of the side chainof the bile acid intermediates di- and trihydroxycoprostanic acids,which results in the formation of the primary bile acids(chenodeoxycholic and cholic acid, respectively), also takes place inperoxisomes. The different fatty acid substrates are likely to bedegraded in distinct beta-oxidation pathways (Baumgart, et al., supra).

Disorders associated with defective peroxisomal fatty acid metabolisminclude adrenoleukodystrophy, adrenomyeloneuropathy, cerebrohepatorenalsyndrome (Zellweger syndrome), Refsum's disease, and peroxisomalthiolase deficiency. Patients with defective peroxisomal fatty acidmetabolism exhibit neuronal demyelination, disordered neuronalmigration, hypotonia, mental retardation, tapetoretinal degeneration,sensorineural hearing loss, cystic changes in the kidneys, skeletalchanges, and death. The clinical distinction between patients with adisorder of peroxisome assembly and those with a defect in a peroxisomalfatty acid metabolic enzyme can be difficult (Watkins, P. A. et al.(1995) Ann. Neurol. 38:472-477).

The discovery of a new human peroxisomal thioesterase and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention and treatmentof cancer, inflammation, and disorders associated with fatty acidmetabolism.

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide, humanperoxisomal thioesterase (PxTE), having the amino acid sequence shown inSEQ ID NO:1, or fragments thereof.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or fragments thereof and a compositioncomprising said polynucleotide sequence. The invention also provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence encoding the amino acid sequence SEQ IDNO:1, or fragments of said polynucleotide sequence. The inventionfurther provides a polynucleotide sequence comprising the complement ofthe polynucleotide sequence encoding the amino acid sequence of SEQ IDNO:1, or fragments or variants of said polynucleotide sequence.

The invention also provides an isolated and purified sequence comprisingSEQ ID NO.2 or variants thereof. In addition, the invention provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of SEQ ID NO:2.

In another aspect the invention provides a composition comprising anisolated and purified polynucleotide sequence comprising the complementof SEQ ID NO:2, or fragments or variants thereof. The invention alsoprovides a polynucleotide sequence comprising the complement of SEQ IDNO:2.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding PxTE under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified PxTE having the amino acid sequence of SEQ IDNO:1 in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist which decreases theactivity of a polypeptide of SEQ ID NO:1. In one aspect the inventionprovides a purified antibody which binds to a polypeptide comprising atleast a fragment of the amino acid sequence of SEQ ID NO:1.

Still further, the invention provides a purified agonist which modulatesthe activity of the polypeptide of SEQ ID NO:1.

The invention also provides a method for treating or preventing adisorder associated with fatty acid metabolism comprising administeringto a subject in need of such treatment an effective amount of apharmaceutical composition comprising purified PxTE.

The invention also provides a method for treating or preventing cancercomprising administering to a subject in need of such treatment aneffective amount of an antagonist of PxTE.

The invention also provides a method for treating or preventinginflammation comprising administering to a subject in need of suchtreatment an effective amount of an antagonist of PxTE.

The invention also provides a method for detecting a polynucleotidewhich encodes PxTE in a biological sample comprising the steps of: a)hybridizing a polynucleotide sequence complementary to PxTE (SEQ IDNO:1) to nucleic acid material of a biological sample, thereby forming ahybridization complex; and b) detecting the hybridization complex,wherein the presence of the complex correlates with the presence of apolynucleotide encoding PxTE in the biological sample. In a preferredembodiment, prior to hybridization, the nucleic acid material of thebiological sample is amplified by the polymerase chain reaction.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of PxTE. The alignment was producedusing MacDNASIS PRO™ software (Hitachi Software Engineering Co. Ltd. SanBruno, Calif.).

FIGS. 2A and 2B show the amino acid sequence alignments among PxTE (SEQID NO:1), TEII from E. coli (GI 147932; SEQ ID NO:3) and CoAthioesterase from yeast (GI 854594; SEQ ID NO:4), produced using themultisequence alignment program of DNASTAR™ software (DNASTAR Inc,Madison Wis.).

FIGS. 3A and 3B show the hydrophobicity plots for PxTE (SEQ ID NO:1) andE. coli TEII (SEQ ID NO:3), respectively; the positive X axis reflectsamino acid position, and the negative Y axis, hydrophobicity (MacDNASISPRO software).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

PxTE, as used herein, refers to the amino acid sequences ofsubstantially purified PxTE obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

The term "agonist", as used herein, refers to a molecule which, whenbound to PxTE, increases or prolongs the duration of the effect of PxTE.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of PxTE.

An "allele" or "allelic sequence", as used herein, is an alternativeform of the gene encoding PxTE. Alleles may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

"Altered" nucleic acid sequences encoding PxTE, as used herein, includethose with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent PxTE. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding PxTE,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding PxTE. The encoded protein may also be "altered" and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent PxTE.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of PxTE is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

"Amino acid sequence", as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of PxTE arepreferably about 5 to about 15 amino acids in length and retain thebiological activity or the immunological activity of PxTE. Where "aminoacid sequence" is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

"Amplification", as used herein, refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "antagonist", as used herein, refers to a molecule which, whenbound to PxTE, decreases the amount or the duration of the effect of thebiological or immunological activity of PxTE. Antagonists may includeproteins, nucleic acids, carbohydrates, or any other molecules whichdecrease the effect of PxTE.

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind PxTE polypeptidescan be prepared using intact polypeptides or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide oroligopeptide used to immunize an animal can be derived from thetranslation of RNA or synthesized chemically and can be conjugated to acarrier protein, if desired. Commonly used carriers that are chemicallycoupled to peptides include bovine serum albumin and thyroglobulin,keyhole limpet hemocyanin. The coupled peptide is then used to immunizethe animal (e.g., a mouse, a rat, or a rabbit).

The term "antigenic determinant", as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term "antisense", as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term "antisense strand" is used in reference toa nucleic acid strand that is complementary to the "sense" strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation "negative" is sometimes used in referenceto the antisense strand, and "positive" is sometimes used in referenceto the sense strand.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic PxTE, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

A "composition comprising a given polynucleotide sequence", as usedherein, refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding PxTE (SEQ ID NO:1) or fragments thereof (e.g., SEQ ID NO:2 andfragments thereof) may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, has been extended usingXL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3' directionand resequenced, or has been assembled from the overlapping sequences ofmore than one Incyte Clone using a computer program for fragmentassembly (e.g., GELVIEW™ Fragment Assembly system, GCG, Madison, Wis.).Some sequences have been both extended and assembled to produce theconsensus sequence.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 by northern analysis is indicative of thepresence of mRNA encoding PxTE in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

A "deletion", as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to PxTE or theencoded PxTE. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term "substantiallyhomologous." The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Human artificial chromosomes (HACs) are linear microchromosomes whichmay contain DNA sequences of 10K to 10M in size and contain all of theelements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

"Microarray" refers to a high-density array of distinct polynucleotidesor oligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

The term "modulate", as used herein, refers to a change in the activityof PxTE. For example, modulation may cause an increase or a decrease inprotein activity, binding characteristics, or any other biological,functional or immunological properties of PxTE.

"Nucleic acid sequence", as used herein, refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. "Fragments" are thosenucleic acid sequences which are greater than 60 nucleotides morepreferably in length, preferably at least 100 nucleotides in length, atleast 1000 nucleotides, in length and most preferably at least 10,000nucleotides in length.

The term "oligonucleotide" refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification, hybridization assays, or microarrays. Asused herein, oligonucleotide is substantially equivalent to the terms"amplimers", "primers", "oligomers", and "probes", as commonly definedin the art.

"Peptide nucleic acid", PNA, as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast five nucleotides in length linked to a peptide backbone of aminoacid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from five amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length PxTE and fragments thereof.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding PxTE, orfragments thereof, or PxTE itself may comprise a bodily fluid, extractfrom a cell, chromosome, organelle, or membrane isolated from a cell, acell, genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport, a tissue, a tissue print, and the like).

The terms "specific binding" or "specifically binding", as used herein,refer to that interaction between a protein or peptide and an agonist,an antibody and an antagonist. The interaction is dependent upon thepresence of a particular structure (i.e., the antigenic determinant orepitope) of the protein recognized by the binding molecule. For example,if an antibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The terms "stringent conditions" or "stringency", as used herein, referto the conditions for hybridization as defined by the nucleic acid,salt, and temperature. These conditions are well known in the art andmay be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

A "variant" of PxTE, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

The Invention

The invention is based on the discovery of a new human peroxisomalthioesterase (hereinafter referred to as "PxTE"), the polynucleotidesencoding PxTE, and the use of these compositions for the diagnosis,prevention, or treatment of cancer, inflammation, and disordersassociated with fatty acid metabolism.

Nucleic acids encoding the PxTE of the present invention were firstidentified in Incyte Clone 2150905 from the fetal brain tissue cDNAlibrary (BRAINOT09) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 1348063 (PROSNOT11), 1506676 (BRAITUT07), 1817644 (PROSNOT20),1931118 (COLNTUT03), 2115316 (BRAITUT03); and GenBank PIDs 1274896 and1313523.

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, and1C. PxTE is 311 amino acids in length and has a peroxisomal targetingsignal at the C-terminus consisting of residues S 309, K 310 and L 311.As shown in FIGS. 2A and 2B, PxTE has chemical and structural homologywith TEII from E. coli (GI 147932; SEQ ID NO:3) and CoA thioesterasefrom yeast (GI 854594; SEQ ID NO:4). In particular, PxTE and E. coliTEII share 44% identity; PxTE and yeast CoA thioesterase share 23%identity. Furthermore, histidine 70 of PxTE aligns with the active-sitehistidine 58 of E. coli TEII. As illustrated by FIGS. 3A and 3B, PxTEand E. coli TEII have rather similar hydrophobicity plots. Northernanalysis shows the expression of this sequence in various libraries,including those prepared from brain and neuronal tissues, colon, smallintestine, lung, pancreas, bladder, prostate, breast, uterus, heart,nasal epithelia, and skin; fetal brain, placenta, and thymus; and celllines derived from promonocytes and mononuclear cells. Of particularnote is the expression of PxTE in fetal and cancer-associated tissues,and tissues associated with inflammation, including Crohn'sdisease-afflicted colon and small intestine, allergy-associatedeosinophilic nasal polyp, and erythema nodosum-afflicted skin tissue.

The invention also encompasses PxTE variants. A preferred PxTE variantis one having at least 80%, and more preferably 90%, amino acid sequenceidentity to the PxTE amino acid sequence (SEQ ID NO:1). A most preferredPxTE variant is one having at least 95% amino acid sequence identity toSEQ ID NO:1.

The invention also encompasses polynucleotides which encode PxTE.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of PxTE can be used to produce recombinant molecules whichexpress PxTE. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in Figs. 1A, 1B, and 1C.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding PxTE, some bearing minimal homology to the nucleotide sequencesof any known and naturally occurring gene, may be produced. Thus, theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequence ofnaturally occurring PxTE, and all such variations are to be consideredas being specifically disclosed.

Although nucleotide sequences which encode PxTE and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring PxTE under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding PxTE or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding PxTE and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode PxTE and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding PxTE or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency as taught in Wahl, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE Amplification Systemmarketed by Gibco/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (PerkinElmer).

The nucleic acid sequences encoding PxTE may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1: 111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5' non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. Genotyper™ and Sequence Navigator™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode PxTE may be used in recombinant DNAmolecules to direct expression of PxTE, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressPxTE.

As will be understood by those of skill in the art, it may beadvantageous to produce PxTE-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter PxTE encodingsequences for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding PxTE may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of PxTE activity, it may be useful toencode a chimeric PxTE protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the PxTE encoding sequence and theheterologous protein sequence, so that PxTE may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding PxTE may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of PxTE, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of PxTE, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active PxTE, the nucleotide sequencesencoding PxTE or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding PxTE andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding PxTE. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene,LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding PxTE,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for PxTE. For example, when largequantities of PxTE are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding PxTE may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding PxTE may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196).

An insect system may also be used to express PxTE. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding PxTE may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of PxTE will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which PxTE may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding PxTE may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing PxTE in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding PxTE. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding PxTE, its initiation codon, and upstream sequences are insertedinto the appropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressPxTE may be transformed using expression vectors which may contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding PxTE isinserted within a marker gene sequence, transformed cells containingsequences encoding PxTE can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding PxTE under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding PxTE and express PxTE may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA--DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding PxTE can be detectedby DNA--DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding PxTE. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding PxTE to detect transformantscontaining DNA or RNA encoding PxTE.

A variety of protocols for detecting and measuring the expression ofPxTE, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson PxTE is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding PxTE includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding PxTE, or anyfragments thereof may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio)).Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding PxTE may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodePxTE may be designed to contain signal sequences which direct secretionof PxTE through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding PxTE to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and PxTE may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingPxTE and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying PxTE from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of PxTE may be producedby direct peptide synthesis using solid-phase techniques Merrifield J.(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer). Various fragments of PxTE may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

Therapeutics

Chemical and structural homology exits among PxTE, TEII from E. coli (GI147932) and CoA thioesterase from yeast (GI 854594). In addition, PxTEis expressed in neuronal, gastrointestinal, and secretory tissues, andcells and tissues associated with inflammation and cancer. Therefore,PxTE appears to play a role in cancer, inflammation, and disordersassociated with fatty acid metabolism.

Therefore, in one embodiment, PxTE or a fragment or derivative thereofmay be administered to a subject to treat a disorder associated withfatty acid metabolism. Such disorders include, but are not limited to,neuronal disorders such as adrenoleukodystrophy, adrenomyeloneuropathy,cerebrohepatorenal syndrome (Zellweger syndrome), Refsum's disease,Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolardisorder, catatonia, dementia, depression, Down's syndrome, tardivedyskinesia, dystonias, epilepsy, Huntington's disease, multiplesclerosis, neurofibromatosis, Parkinson's disease, paranoid psychoses,schizophrenia, and Tourette's disorder.

In another embodiment, a vector capable of expressing PxTE, or afragment or a derivative thereof, may also be administered to a subjectto treat a disorder associated with fatty acid metabolism including, butnot limited to those described above.

In still another embodiment, an agonist of PxTE may also be administeredto a subject to treat a disorder associated with fatty acid metabolismincluding, but not limited to those described above.

In one embodiment, an antagonist of PxTE may be administered to asubject to prevent or treat cancer. Such cancers may include, but arenot limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, and teratocarcinoma, and particularly cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In one aspect, antibodieswhich specifically bind PxTE may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express PxTE.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding PxTE may be administered to a subject to treator prevent cancer including, but not limited to those described above.

In one embodiment, an antagonist of PxTE may be administered to asubject to prevent or treat inflammation. Inflammation may result fromany condition or disorder and in particular, conditions or disorderswhich include but are not limited to Addison's disease, adultrespiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves'disease, hypereosinophilia, irritable bowel syndrome, lupuserythematosus, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma. In one aspect,antibodies which specifically bind PxTE may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express PxTE.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding PxTE may be administered to a subject to treator prevent inflammation associated with any condition or disorder andincluding, but not limited to the disorders described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

Antagonists or inhibitors of PxTE may be produced using methods whichare generally known in the art. In particular, purified PxTE may be usedto produce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind PxTE.

Antibodies to PxTE may be generated using methods that are well known inthe art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith PxTE or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to PxTE have an amino acid sequence consisting of atleast five amino acids and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of PxTE amino acids may be fused with those of another proteinsuch as keyhole limpet hemocyanin and antibody produced against thechimeric molecule.

Monoclonal antibodies to PxTE may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to producePxTE-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Nati. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. NatI. Acad. Sci. 86: 3833-3837; Winter,G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for PxTE mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between PxTE and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering PxTE epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingPxTE, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingPxTE may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding PxTE. Thus,complementary molecules or fragments may be used to modulate PxTEactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments, can be designed from various locations along thecoding or control regions of sequences encoding PxTE.

Expression vectors derived from retro viruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encoding PxTE.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

Genes encoding PxTE can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes PxTE. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5' or regulatory regions of the gene encoding PxTE(signal sequence, promoters, enhancers, and introns). Oligonucleotidesderived from the transcription initiation site, e.g., between positions-10 and +10 from the start site, are preferred. Similarly, inhibitioncan be achieved using "triple helix" base-pairing methodology. Triplehelix pairing is useful because it causes inhibition of the ability ofthe double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Thecomplementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding PxTE.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding PxTE. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections orpolycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of PxTE, antibodies toPxTE, mimetics, agonists, antagonists, or inhibitors of PxTE. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of PxTE, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example PxTE or fragments thereof, antibodies of PxTE,agonists, antagonists or inhibitors of PxTE, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind PxTE may beused for the diagnosis of conditions or diseases characterized byexpression of PxTE, or in assays to monitor patients being treated withPxTE, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for PxTE includemethods which utilize the antibody and a label to detect PxTE in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuring PxTEare known in the art and provide a basis for diagnosing altered orabnormal levels of PxTE expression. Normal or standard values for PxTEexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toPxTE under conditions suitable for complex formation The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric, means. Quantities of PxTE expressed insubject samples, control and disease from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingPxTE may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofPxTE may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of PxTE,and to monitor regulation of PxTE levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding PxTE or closely related molecules, may be used to identifynucleic acid sequences which encode PxTE. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding PxTE, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe PxTE encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring PxTE.

Means for producing specific hybridization probes for DNAs encoding PxTEinclude the cloning of nucleic acid sequences encoding PxTE or PxTEderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding PxTE may be used for the diagnosis ofconditions, disorders, or diseases which are associated with expressionof PxTE. Examples of such conditions or diseases includeadrenoleukodystrophy, adrenomyeloneuropathy, cerebrohepatorenal syndrome(Zellweger syndrome), Refsum's disease, Alzheimer's disease, amnesia,amyotrophic lateral sclerosis, bipolar disorder, catatonia, dementia,depression, Down's syndrome, tardive dyskinesia, dystonias, epilepsy,Huntington's disease, multiple sclerosis, neurofibromatosis, Parkinson'sdisease, paranoid psychoses, schizophrenia, and Tourette's disorder;adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma, and particularly cancers of the adrenal gland, bladder,bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen,testis, thymus, thyroid, and uterus; Addison's disease, adultrespiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma. The polynucleotidesequences encoding PxTE may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; orin dipstick, pin, ELISA assays or microarrays utilizing fluids ortissues from patient biopsies to detect altered PxTE expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding PxTE may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingPxTE may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding PxTE in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of PxTE, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes PxTE, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding PxTE may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5'→3') and another with antisense (3'←5'),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

Methods which may also be used to quantitate the expression of PxTEinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation.

In further embodiments, oligonucleotides derived from any of thepolynucleotide sequences described herein may be used as probes inmicroarrays. The microarrays can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents.

In one embodiment, the microarray is prepared and used according to themethods described in PCT application WO95/11995 (Chee et al.), Lockhart,D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al.(1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which areincorporated herein in their entirety by reference.

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs fixed to a solidsupport. Microarrays may contain pairs of oligonucleotides which coverthe known 5', or 3', sequence, or contain sequential oligonucleotideswhich cover the full length sequence; or unique oligonucleotidesselected from particular areas along the length of the sequence. The"pairs" will consist of two strands which are identical except for onenucleotide, preferably in the center. The number of oligonucleotidepairs may range from 10-500 for a given sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5' or more preferably at the 3' end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. The oligomers are synthesized atdesignated areas on a substrate using a light-directed chemical process.The substrate may be paper, nylon or other type of membrane, filter,chip, glass slide or any other suitable solid support.

In another aspect, the oligonucleotides may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a "gridded" array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available devises (slot blot or dot blotapparatus) materials and machines (including robotic instruments) andcontain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144dots, or any other multiple which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using the microarrays, the RNA orDNA from a biological sample is made into hybridization probes. The mRNAis isolated, and cDNA is produced and used as a template to makeantisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences, mutations, variants, or polymorphisms amongsamples.

In another embodiment of the invention, the nucleic acid sequences whichencode PxTE may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome or to artificial chromosome constructions, such as humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions orsingle chromosome cDNA libraries as reviewed in Price, C. M. (1993)Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

Fluorescent in situ hybridization (FISH as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in various scientific journals or at Online Mendelian Inheritancein Man (OMIM). Correlation between the location of the gene encodingPxTE on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, PxTE, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenPxTE and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to PxTE large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with PxTE, or fragments thereof, and washed. Bound PxTE is thendetected by methods well known in the art. Purified PxTE can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding PxTE specificallycompete with a test compound for binding PxTE. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with PxTE.

In additional embodiments, the nucleotide sequences which encode PxTEmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I BRAINOT09 cDNA Library Construction

The BRAINOT09 cDNA library was constructed from microscopically normalfetal brain tissue obtained from a Caucasian male (specimen#RU95-10-0700; International Institute for the Advancement of Medicine,Exton, Pa.) who died after 23 weeks' gestation following prematurebirth.

The frozen tissue was homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, N.Y.) inguanidinium isothiocyanate solution. The lysate was extracted once withacid phenol at pH 4.7 per Stratagene's RNA isolation protocol(Stratagene, Inc., San Diego, Calif.). The RNA was extracted once withan equal volume of acid phenol, reprecipitated using 0.3M sodium acetateand 2.5 volumes of ethanol, resuspended in DEPC-treated water, and DNasetreated for 25 min at 37° C. The RNA extraction and precipitation wererepeated as before. The mRNA was isolated with the Qiagen Oligotex kit(QIAGEN, Inc.; Chatsworth, Calif.) and used to construct the cDNAlibrary.

The mRNA was handled according to the recommended protocols in theSuperScript Plasmid System (Cat. #18248-013; Gibco/BRL). The cDNAs werefractionated on a Sepharose CL4B column (Cat. #275105-01; Pharmacia),and those cDNAs exceeding 400 bp were ligated into pINCY I. The plasmidpINCY I was subsequently transformed into DH5a™ competent cells (Cat.#18258-012; Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 Plasmid Kit (Catalog #26173; QIAGEN, Inc.). The recommended protocolwas employed except for the following changes: 1) the bacteria werecultured in 1 ml of sterile Terrific Broth (Catalog #22711, Gibco/BRL)with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) afterinoculation, the cultures were incubated for 19 hours and at the end ofincubation, the cells were lysed with 0.3 ml of lysis buffer; and 3)following isopropanol precipitation, the plasmid DNA pellet wasresuspended in 0.1 ml of distilled water. After the last step in theprotocol, samples were transferred to a 96-well block for storage at 4°C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.)in combination with Peltier Thermal Cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing Systems.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences of the Sequence Listing or amino acid sequencesdeduced from them were used as query sequences against databases such asGenBank, SwissProt, BLOCKS, and Pima II. These databases which containpreviously identified and annotated sequences were searched for regionsof homology (similarity) using BLAST, which stands for Basic LocalAlignment Search Tool (Altschul, S. F. (1993) J. Mol. Evol. 36:290-300;Altschul et al. (1990) J. Mol. Biol. 215:403-410).

BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal or plant) origin. Other algorithms such asthe one described in Smith R. F. and T. F. Smith (1992; ProteinEngineering 5:35-51), incorporated herein by reference, can be used whendealing with primary sequence patterns and secondary structure gappenalties. As disclosed in this application, the sequences have lengthsof at least 49 nucleotides, and no more than 12% uncalled bases (where Nis recorded rather than A, C, G, or T).

The BLAST approach, as detailed in Karlin, S. and S. F. Altschul (1993;Proc. Nat. Acad. Sci. 90:5873-7) and incorporated herein by reference,searches for matches between a query sequence and a database sequence,to evaluate the statistical significance of any matches found, and toreport only those matches which satisfy the user-selected threshold ofsignificance. In this application, threshold was set at 10⁻²⁵ fornucleotides and 10⁻¹⁴ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and mammalian sequences (mam), anddeduced amino acid sequences from the same clones are searched againstGenBank functional protein databases, mammalian (mamp), vertebrate(vrtp) and eukaryote (eukp), for homology. The relevant database for aparticular match were reported as a GIxxx±p (where xxx is pri, rod, etcand if present, p=peptide).

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:

    % sequence identity×% maximum BLAST score/100

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1-2% error;and at 70, the match will be exact. Homologous molecules are usuallyidentified by selecting those which show product scores between 15 and40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding PxTE occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of PxTE Encoding Polynucleotides

The nucleic acid sequence of the Incyte Clone 2150905 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension in theantisense direction, and the other was synthesized to extend sequence inthe sense direction. Primers were used to facilitate the extension ofthe known sequence "outward" generating amplicons containing new,unknown nucleotide sequence for the region of interest. The initialprimers were designed from the cDNA using OLIGO 4.06 (NationalBiosciences), or another appropriate program, to be about 22 to about 30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures of about 68° to about 72°C. Any stretch of nucleotides which would result in hairpin structuresand primer-primer dimerizations was avoided.

Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. Beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, PCR was performedusing the Peltier Thermal Cycler (PTC200; M. J. Research, Watertown,Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                   Step 2       65° C. for 1 min                                          Step 3       68° C. for 6 min                                          Step 4       94° C. for 15 sec                                         Step 5       65° C. for 1 min                                          Step 6       68° C. for 7 min                                          Step 7       Repeat step 4-6 for 15 additional cycles                         Step 8       94° C. for 15 sec                                         Step 9       65° C. for 1 min                                          Step 10      68° C. for 7:15 min                                       Step 11      Repeat step 8-10 for 12 cycles                                   Step 12      72° C. for 8 min                                          Step 13      4° C. (and holding)                                       ______________________________________                                    

A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQuick™ (QIAGEN Inc., Chatsworth, Calif.), and trimmedof overhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2× Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2× Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                           Step 2     94° C. for 20 sec                                           Step 3     55° C. for 30 sec                                           Step 4     72° C. for 90 sec                                           Step 5     Repeat steps 2-4 for an additional 29 cycles                       Step 6     72° C. for 180 sec                                          Step 7     4° C. (and holding)                                         ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5' regulatory sequences using the procedure above, oligonucleotidesdesigned for 5' extension, and an appropriate genomic library.

VI Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of γ-³² p! adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1,or Pvu II; DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Microarrays

To produce oligonucleotides for a microarray, the nucleotide sequencedescribed herein is examined using a computer algorithm which starts atthe 3' end of the nucleotide sequence. The algorithm identifiesoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that would interfere with hybridization. Thealgorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25 116, incorporated herein byreference). In another alternative, a "gridded" array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

VIII Complementary Polynucleotides

Sequence complementary to the PxTE-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring PxTE. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of PxTE, SEQ ID NO:1. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5' sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the PxTE-encoding transcript.

IX Expression of PxTE

Expression of PxTE is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express PxTE in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofPxTE into the bacterial growth media which can be used directly in thefollowing assay for activity.

X Demonstration of PxTE Activity

The thioesterase activity of PxTE is assayed by monitoring theappearance of the CoA-thiol hydrolysis product. Incubations contain 0.1mM 5,5'-dithiobis(2-nitrobenzoate), 20 μM fatty acyl-CoA (such asdecanoyl-CoA), and 0.1M phosphate buffer pH 7.5 at 37° C. Reactions areinitiated by adding PxTE and monitored spectrophotometrically byrecording the increase in absorbance at 412 nm.

XI Production of PxTE Specific Antibodies

PxTE that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regions,is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio iodinated, goat anti-rabbitIgG.

XII Purification of Naturally Occurring PxTE Using Specific Antibodies

Naturally occurring or recombinant PxTE is substantially purified byimmunoaffinity chromatography using antibodies specific for PxTE. Animmunoaffinity column is constructed by covalently coupling PxTEantibody to an activated chromatographic resin, such as CNBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing PxTE is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof PxTE (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/PxTE binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and PxTE is collected.

XIII Identification of Molecules Which Interact with PxTE

PxTE or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled PxTE, washed and any wells withlabeled PxTE complex are assayed. Data obtained using differentconcentrations of PxTE are used to calculate values for the number,affinity, and association of PxTE with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 311 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: BRAINOT09                                                        (B) CLONE: 2150905                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetGlyArgAlaValAlaThrAlaAlaLeuProProGlyAspLeuArg                              151015                                                                        SerValLeuValThrThrValLeuAsnLeuGluProLeuAspGluAsp                              202530                                                                        LeuPheArgGlyArgHisTyrTrpValProAlaLysArgLeuPheGly                              354045                                                                        GlyGlnIleValGlyGlnAlaLeuValAlaAlaAlaLysSerValSer                              505560                                                                        GluAspValHisValHisSerLeuHisCysTyrPheValArgAlaGly                              65707580                                                                      AspProLysLeuProValLeuTyrGlnValGluArgThrArgThrGly                              859095                                                                        SerSerPheSerValArgSerValLysAlaValGlnHisGlyLysPro                              100105110                                                                     IlePheIleCysGlnAlaSerPheGlnGlnAlaGlnProSerProMet                              115120125                                                                     GlnHisGlnPheSerMetProThrValProProProGluGluLeuLeu                              130135140                                                                     AspCysGluThrLeuIleAspGlnTyrLeuArgAspProAsnLeuGln                              145150155160                                                                  LysArgTyrProLeuAlaLeuAsnArgIleAlaAlaGlnGluValPro                              165170175                                                                     IleGluIleLysProValAsnProSerProLeuSerGlnLeuGlnArg                              180185190                                                                     MetGluProLysGlnMetPheTrpValArgAlaArgGlyTyrIleGly                              195200205                                                                     GluGlyAspMetLysMetHisCysCysValAlaAlaTyrIleSerAsp                              210215220                                                                     TyrAlaPheLeuGlyThrAlaLeuLeuProHisGlnTrpGlnHisLys                              225230235240                                                                  ValHisPheMetValSerLeuAspHisSerMetTrpPheHisAlaPro                              245250255                                                                     PheArgAlaAspHisTrpMetLeuTyrGluCysGluSerProTrpAla                              260265270                                                                     GlyGlySerArgGlyLeuValHisGlyArgLeuTrpArgGlnAspGly                              275280285                                                                     ValLeuAlaValThrCysAlaGlnGluGlyValIleArgValLysPro                              290295300                                                                     GlnValSerGluSerLysLeu                                                         305310                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1098 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: BRAINOT09                                                        (B) CLONE: 2150905                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CAGCATTGAACTAGATGTCGTCCCCGCAGGCCCCAGAAGATGGGCAGGGCTGTGGCGACC60                GCGGCGCTTCCCCCTGGGGACCTCCGTAGCGTCTTGGTCACGACCGTGCTCAACCTCGAG120               CCGCTGGACGAGGATCTCTTCAGAGGAAGGCATTACTGGGTACCGGCCAAGAGGCTGTTT180               GGTGGTCAGATCGTGGGCCAGGCCCTGGTGGCTGCAGCCAAGTCTGTGAGTGAAGACGTC240               CACGTGCACTCCCTGCACTGCTACTTTGTTCGGGCAGGGGACCCGAAGCTGCCAGTACTG300               TACCAAGTGGAGCGGACACGAACAGGGTCGAGCTTCTCGGTGCGCTCTGTGAAGGCCGTG360               CAACATGGGAAGCCCATCTTCATCTGCCAGGCCTCCTTCCAGCAGGCCCAGCCCAGCCCC420               ATGCAGCACCAGTTCTCCATGCCCACTGTGCCACCACCAGAAGAGCTGCTTGACTGTGAG480               ACCCTCATTGACCAGTATTTAAGGGACCCTAACCTCCAAAAGAGGTACCCATTGGCGCTC540               AACCGAATTGCTGCTCAGGAGGTCCCCATTGAGATCAAGCCAGTAAACCCATCCCCCCTG600               AGCCAGCTGCAGAGAATGGAGCCCAAACAGATGTTCTGGGTGCGAGCCCGGGGCTATATT660               GGCGAGGGCGACATGAAGATGCACTGCTGCGTGGCCGCCTATATCTCCGACTATGCCTTC720               TTGGGCACTGCACTGCTGCCTCACCAGTGGCAGCACAAGGTGCACTTCATGGTCTCACTG780               GACCATTCCATGTGGTTCCACGCCCCCTTCCGAGCTGACCACTGGATGCTCTATGAATGC840               GAGAGCCCCTGGGCCGGTGGCTCTCGGGGGCTGGTCCATGGGCGGCTGTGGCGTCAGGAT900               GGAGTCCTAGCTGTGACCTGTGCCCAGGAGGGCGTGATCCGAGTGAAGCCCCAGGTCTCA960               GAGAGCAAGCTGTAGCCAGAGGTACCAGCTTCGCCTGGGGCTTCAAGAACCTCCCATCTA1020              TCCCCATTCCTGAGACAGGAGTTACAGTCCCTTTTGGCCCTCACATCCAATAAAGAGACT1080              GATACCACTGGAAAAAAA1098                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 286 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 147932                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetSerGlnAlaLeuLysAsnLeuLeuThrLeuLeuAsnLeuGluLys                              151015                                                                        IleGluGluGlyLeuPheArgGlyGlnSerGluAspLeuGlyLeuArg                              202530                                                                        GlnValPheGlyGlyGlnValValGlyGlnAlaLeuTyrAlaAlaLys                              354045                                                                        GluThrValProGluGluArgLeuValHisSerPheHisSerTyrPhe                              505560                                                                        LeuArgProGlyAspSerLysLysProIleIleTyrAspValGluThr                              65707580                                                                      LeuArgAspGlyAsnSerPheSerAlaArgArgValAlaAlaIleGln                              859095                                                                        AsnGlyLysProIlePheTyrMetThrAlaSerPheGlnAlaProGlu                              100105110                                                                     AlaGlyPheGluHisGlnLysThrMetProSerAlaProAlaProAsp                              115120125                                                                     GlyLeuProSerGluThrGlnIleAlaGlnSerLeuAlaHisLeuLeu                              130135140                                                                     ProProValLeuLysAspLysPheIleCysAspArgProLeuGluVal                              145150155160                                                                  ArgProValGluPheHisAsnProLeuLysGlyHisValAlaGluPro                              165170175                                                                     HisArgGlnValTrpIleArgAlaAsnGlySerValProAspAspLeu                              180185190                                                                     ArgValHisGlnTyrLeuLeuGlyTyrAlaSerAspLeuAsnPheLeu                              195200205                                                                     ProValAlaLeuGlnProHisGlyIleGlyPheLeuGluProGlyIle                              210215220                                                                     GlnIleAlaThrIleAspHisSerMetTrpPheHisArgProPheAsn                              225230235240                                                                  LeuAsnGluTrpLeuLeuTyrSerValGluSerThrSerAlaSerSer                              245250255                                                                     AlaArgGlyPheValArgGlyGluPheTyrThrGlnAspGlyValLeu                              260265270                                                                     ValAlaSerThrValGlnGluGlyValMetArgAsnHisAsn                                    275280285                                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 349 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 854594                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetSerAlaSerLysMetAlaMetSerAsnLeuGluLysIleLeuGlu                              151015                                                                        LeuValProLeuSerProThrSerPheValThrLysTyrLeuProAla                              202530                                                                        AlaProValGlySerLysGlyThrPheGlyGlyThrLeuValSerGln                              354045                                                                        SerLeuLeuAlaSerLeuHisThrValProLeuAsnPhePheProThr                              505560                                                                        SerLeuHisSerTyrPheIleLysGlyGlyAspProArgThrLysIle                              65707580                                                                      ThrTyrHisValGlnAsnLeuArgAsnGlyArgAsnPheIleHisLys                              859095                                                                        GlnValSerAlaTyrGlnHisAspLysLeuIlePheThrSerMetIle                              100105110                                                                     LeuPheAlaValGlnArgSerLysGluHisAspSerLeuGlnHisTrp                              115120125                                                                     GluThrIleProGlyLeuGlnGlyLysGlnProAspProHisArgTyr                              130135140                                                                     GluGluAlaThrSerLeuPheGlnLysGluValLeuAspProGlnLys                              145150155160                                                                  LeuSerArgTyrAlaSerLeuSerAspArgPheGlnAspAlaThrSer                              165170175                                                                     MetSerLysTyrValAspAlaPheGlnTyrGlyValMetGluTyrGln                              180185190                                                                     PheProLysAspMetPheTyrSerAlaArgHisThrAspGluLeuAsp                              195200205                                                                     TyrPheValLysValArgProProIleThrThrValGluHisAlaGly                              210215220                                                                     AspGluSerSerLeuHisLysHisHisProTyrArgIleProLysSer                              225230235240                                                                  IleThrProGluAsnAspAlaArgTyrAsnTyrValAlaPheAlaTyr                              245250255                                                                     LeuSerAspSerTyrLeuLeuLeuThrIleProTyrPheHisAsnLeu                              260265270                                                                     ProLeuTyrCysHisSerPheSerValSerLeuAspHisThrIleTyr                              275280285                                                                     PheHisGlnLeuProHisValAsnAsnTrpIleTyrLeuLysIleSer                              290295300                                                                     AsnProArgSerHisTrpAspLysHisLeuValGlnGlyLysTyrPhe                              305310315320                                                                  AspThrGlnSerGlyArgIleMetAlaSerValSerGlnGluGlyTyr                              325330335                                                                     ValValTyrGlySerGluArgAspIleArgAlaLysPhe                                       340345                                                                        __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide sequencewhich encodes the peroxisomal thioesterase of SEQ ID NO:1 or a variantthereof which differs by one amino acid and retains enzymatic activity.2. A composition comprising the polynucleotide sequence of claim
 1. 3. Apolynucleotide which is complementary to the polynucleotide of claim 1.4. An isolated and purified polynucleotide sequence comprising SEQ IDNO:2.
 5. A composition comprising the polynucleotide sequence of claim4.
 6. A polynucleotide sequence which is complementary to thepolynucleotide sequence of claim
 4. 7. An expression vector containingthe polynucleotide of claim
 2. 8. A host cell containing the vector ofclaim
 7. 9. A method for producing a polypeptide comprising the aminoacid sequence of SEQ ID NO:1, the method comprising the steps of:a)culturing the host cell of claim 8 under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.